title: Mode of binding of RNA polymerase α subunit to the phased A-tracts upstream of the phospholipase C gene promoter of Clostridium perfringens.
abstract: Three phased A5-6-tracts lie upstream of the promoter of plc encoding the α-toxin (phospholipase C) of Clostridium perfringens. The α subunits of C. perfringens RNA polymerase bind directly to the phased A-tracts via the C-terminal domain of the α subunit (αCTD). To identify the amino acid residues involved in the binding of C. perfringens α subunits to the phased A-tracts, 27 amino acid residues in C. perfringens αCTD were substituted with alanine. The affinities of the mutated α subunits for the phased A-tracts were examined by gel shift assays and surface plasmon resonance (SPR). The SPR analyses revealed that the phased A-tracts themselves facilitated a complex formation between the phased A-tracts and C. perfringens α subunits [Kd was 6.1 (±0.3) × 10(-8) M], and that Arg261, Asn264, Gly292 and Lys294 in C. perfringens αCTD were critical for the binding to the phased A-tracts. The topology of these amino acid residues on the predicted structure of C. perfringens αCTD indicated a contact path with the phased A-tracts that was similar to that of Escherichia coli αCTD with the upstream (UP) element. On the other hand, SPR analyses at different temperatures (15, 25 and 37 °C) indicated that the affinity of the C. perfringens α subunits for the phased A-tracts increased in a low-temperature-dependent manner, whereas that of the E. coli α subunit for the UP element did not. This suggests that the phased A-tracts may not simply be a subset of the UP element, and that they show specific binding activity with the RNA polymerase α subunit.
authors.affiliation: Department of Life Science, Faculty of Science, Okayama University of Science, 1-1, Ridai-cho, Okayama 700-0005, Japan. Electronic address: katayama@dls.ous.ac.jp.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Katayama
authors.forename: Kotaro
authors.initials: K
authors.lastname: Ishibashi
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Gotoh
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nakamura
mesh_terms: D019943:Amino Acid Substitution; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance; D013696:Temperature; D010738:Type C Phospholipases / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit; D010738:Type C Phospholipases
keywords: Clostridium perfringens; Phased A-tracts; Surface plasmon resonance (SPR); UP element; α subunit of RNA polymerase; α-toxin (phospholipase C)